sUTES
July 1969
ti97
TABLE I
Yield, Cor11p11
It 1
It 2
CH;
CH;
R3
%
R4
-Mp, Base
OC--
HCI
Formulae
Re1 act./
1 2
a
4 6 6 7 8
9 10 11
12 13 14 15 16 17 18 19 20 a Reference 4 ; melting point was not given. * Reference 2 ; melting point was not given. c Lit.j mp 175-177'. d Reference 3 ; melting point was not given. e All compounds prepared were subjeckd to ir and nmr spectroscopy and showed the expected absorptions. Analytical data were within 10.307, of theoretical values. f Procaine hydrochloride as a 1%solution by subcut,aneoas injection = 1; - = inactive. SCHEME I
CIQS+~H>
----+ KMnO,
-
N ~ O , S ~ C O , HSO H CI
Na0H
-
R 1 > N O , s ~ c o ~ SOCI, H
to react 1 hr at room temoerature, then excess SOCL was distilled off and the residue was washed with ice-water and filtered until dry. The yield was almost quantitative, mp 154". dnal. (CiiHizClX0,S) C1. $-Pyrrolidinosulfonylbenzoic Acid Dimethylaminoethyl Ester (13).-To p-pyrrolidinosulfonylbenzoyl chloride (2.7 g, 0.01 mole) in 20 ml of dry CGHB was added 0.9 g (0.01 mole) of diThe mixture methylaminoethyl alcohol in 5 ml of dry C&. was allowed to stand overnight a t room temperature and then the solvent was evaporated in vacuo. The crystalline mass was recrystallized (EtOAc-EtOH) to give 2.3 g (657,) of product, mp 203". The free basewas recrystallized from EtOH-HzO; m p 8 5 O , ir and nmr (CDC13) as expected. A4nal. See Table I.
R,
Inhibition of Cholesterolgenesis i n Vitro by Chlorophenoxyacetic Acids. Effect of a-Methyl G r o u p s 1 8
chloride. The corresponding piperidinoethyl ester (11) was as active as cocaine hydrochloride. Subcutaneous injections of 0.5 ml of 1% solutions of 9 or 11 showed an activity comparable with that of 0.5 ml of 0.5Y0 of procaine hydrochloride. The local anesthetic activity of the compounds prepared are included in Table I. Experimental Section'
$-Pyrrolidinosulfonylbenzoic Acid.-To
a solution of 7.1 g (0.1 mole) of pyrrolidine in 40 ml of 107, aqueous NaOH was added with stirring 11 g (0.05 mole) of p-chlorosulfonylbenzoic acid.8 Bfter 3 hr the clear solution &'as acidified wit,h HC1 and the precipitate w-as filtered and recrystallized from AIeOH-;CIezCO to give 6 g (56%) of product, mp 239", ir and nnir (CRCOOH) as expected. A n a l . (C11H1aX04S)N. $-Pyrrolidinosulfonylbenzoyl Chloride.-p-Pyrrolidinosidfonylbenzoic acid ( 2 . 5 5 g 0.01 mole), and 5 ml of SOC1, were allowed (7) Melting points were taken on a Kofler hot stage microscope. The ir spectra were determined with FL Leitz Model 111 spectrograph (KBr). N m r spectra were obtained on a Varian A60h instrument using MerSi as internal standard. (8) S. Sniiles and 0. C. Harrison, J . Chem. Soc., 121, 2022 (1922).
DONALD T . WITIAK,ROBERT E . HACKNEY,^^ AXD ~ I I C H A E W.L WHITEHOUSE~C Division of Medicinal Chemistry, College of Pharmacy, The Ohio State Cniversity, Columbus, Ohio 43210 Received IMarch 10, 1969
We recently reported that the two isomeric desmethyl analogs of ethyl a-(4-chlorophenoxy)-a-methylpropionate (1) differ in their hypocholesterolemic activity in rats; only the L isomer 2 is equally as active as 1 in The D isomer 2 is not significantly active. Experiments in this laboratory have shown that like 1, which readily undergoes in uivo and in vitro hydrolysis to 3, esters L-2and D-2 are readily hydrolyzed t o their respective acids L-4 and n-4 by rat liver, serum, and (1) (a) Presented t o t h e Division of Medicinal Chemistry, 157th Xational Meeting of the American Chemical Society, Minneapolis. XIinn., April 13-18, 1969. (b) .-imerican Foundation for Pharmaceutical Education Fellow. (c) Sational Science Foundation Senior Visiting Foreign Scientist 19671968. (2) Clofibrate: clilorpenisate, ICI-28257. CPII3. .\tromid-S, Regelan. (3) D. T . Witiak, T. C.-L. Ho, R . E. Hackney. and TF-. E. Connor, J . M e d . Chem., 11, 1086 (1968).
iutestirial preparation^.^ The acid 3 is Itriown to blocl\ sterol biosynthesis after mevalonatej and before squalene6 with a minimal effect before mevalon:ite.b Yo1 these reasons \le have now examined the effects of 3, 1,-4, D-4, and 5, added iu i ~ i t ) ooii , the incorjmxtioii ol :ice1 :it e-]- l 4 c ' :md nicvuloiiat ~ - 2 - ~ ~iut( 0' ~ i o i i ~ i i p o r i i fiahle materiul, squalene aiitl cholrbtt~rol.
1,R = C2H3,R=H
I.-2.R = CIHi I -4. R H
Figure 1.--l~ose-i,uapoiize ctirves Tor t,he per ceiii, inhibitiori of incorporation of mevalonate-2-14C into nonsaporiifiable material in fortified rat liver homogenatrs: ( A j n-(4-chlorophenoxy)-aniethylpropionic acid (3), ( R ) I,-( - j-a-(4-~hlorophenosy)propionir acid (4), i C ) n-i )-or-(4-chlorophenoxy)propionic arid (4), (1) ) ~u-(4-~hloroptiriios3.)acetic acid ( 5 ) .
+
D-2,R
CjH; n.4.R = H
5
The Inhibitory effect of these compounds 011 the illcorporation of acetate-l-'*C into rionsnpoiiifiable material is shoirti i i i Tablc I. At :i coiiceiitrntioii of l.: mall acid', 3, I,-4, :md 5 irihibitetl acet:ite iricorpor:ttiori about :3OC,&. T h e inhibition :it the 1.3-mJf dose \vas glitly grc:iter \T lieii acetate rather thaii mevaloriate i g i m 1) wn.: iiicd, but with either precursor the difference in :tctivity hetween 3, L-4, :tnd 5 wis not sigiiificaiit. The 1)-4 iwnier exhibited le's than one-fourth the activity (1' < 0.02) of either 3, L-4, or 5 when acetate u :is the prerimor. Dost. -rehpoiise curves (Figurcb 1) further subbtantinte the greater i)i u f i o activity of acids 3, L-4, mid 5 . \J7ithin experimental error these three compounds exhibited essentially the same c u r w with approximately 90% inhibition of incorporation of r t i e ~ , ~ ~ l o i i a t e - 2into - 1 ~ ~nonsaporiifiable material a t :i conccntrxtioii of 9 mJ1. -It this coiiccAiitration u-4 irihibitcd mcvaloiiatc incorporation iiot more than 205; .
Experimental Section
C'ombinatioiis of either 1.3 mJ13 plus 1.3 n i M 1,-4 or l..j m X 3 plui 1.5 m J 1 5 gare the same per cent inhihit ion of incorporation of mevalo1iate-2--'~Cinto non*:iponifiable material ab did 3 n i J l 3 alone; i.e.. no qyriergistic eflects are observed. Had 3 synergistic cff'ect bceri found, this might have suggested two different sites in the over-all pathway of sterolgenesis for the action of L-4 arid 3 or 5. .Iiidysis hy gas-liquid partition chromatography (sec Experimental Section) indicated that cholesterol :iccuuntcd for approximately 8.3%:: of the rionsapoiiifi-
~~-(4-Chlorophenoxyj-~-methylpropionic acid (3), I,-( - )-a-[4chlorophenoxyjpropionic acid (4), and I)-( f ) - ~ ( l - c h l o r o p h e n 0xy)propionic acid ( 4 ) were syiitheaized arcordixig t o nicthotis previoidy piiblishetl.~ 4-Chlorophenoxyacetic acid ( 5 ) \?:Inl:i1i Kodak Co,, Rochestcr, N . Y. ~ I I I -4, aiid 1)-4 \?ere prepared by diwdvhig l l i ( t ertly i i i 0.1 .I[ phosphate t)iifYer>pT-37.4. Biological.--Liver,5 were o1)t aiiird i ~ healthy ~ m n i d e \\'i>t t'r rat3 (200-230 a). 1,iver horiiogeiiates were prepared by Uiicthrl,'s methods and debri, \vaA i~eniovedby slow centrifugation (3OOy, 2..i Inin). Oiie liver (10 g) yielded approximately 20 nil of hitpernatant. Each iiiciiht i o i i flask contained 2 rill of superliatant, pCi of acetate-l-lT, 0 . t 1111 of st,ook drug solkitioiis, aiid factorsg wit,h added strdillrn citrate (final concentratiori 2 r r i nmde up to 4.0 ml with 0.I .\I phosphate buffer, pH 7.4. In order to accommodate B greater volume of 50 m M drug solution for dose-response studies wlieii irievalunat,e-'2-'4C (0.23 pcli) wns em.
.-
( 7 ) I ) . I t , . ~ Y O , \ , I:. .\. . $ t , > - j > d , a i d I ? , G , Guuld, J . Lipid K c s . , 6 , :l(i!l ( 196.5), 18) X. L. 1%.13uch?r. ./. . I u I . Ciicm. Sor... 1 5 , 1!)8 (1953). (Y) W. L.I l o l m w a n d .I. 1). Uentz. J . B i d . Chem.,236, 3118 f 1 M U j .
NOTES
July 1969
699
ployed, 1.5 ml of supernatant was utilized. Concentrations of all cofactors remained the same and the final volume was similarly made up to 4.0 ml with 0.1 M phosphate buffer. After 1 hr of incubation in air with slow shaking a t 37" the reaction was stopped by adding 3.0 ml of 167, KOH in 50% EtOH followed by heating a t 65-70' in a water bath for 10 min. The partially saponified reaction mixture was transferred t'o a 20-ml glassstoppered centrifuge tube and the flask was rinsed with 3.0 ml of the alcoholic KOH solution which was added to the contents of the centrifuge tube. Saponification was completed by heating the tightly stoppered tube in a water bath (75-80') for 1hr. The saponified mixtures were extracted three times with 5.0-ml portions of petroleum ether (bp 60-80"). The combined extracts were diluted t o 25.0 ml and dried (Xa2SO4). Five milliliters of the dried petroleum ether extract was diluted with 10.0 ml of PPO (2,5-diphenyloxaxole) solution (0.4% PPO in Ph&le-95% EtOH, 70: 30 v/v) in standard counting vials. The radioactivity was determined with a Packard Tri-Carb liquid scintillation counter. A sufficient number of counts was taken to reduce the statistical error of counting to less than 5%. I n each run, as a further check on the extraction procedure (and as a measure of possible contamination), the contents of one incubation flask was treated with KOH solution prior t o incubation; the radioactivity in the petroleum ether extract derived from these alkali pretreated incubations was always found to be negligible. Gas-liquid partition chromatography10of the nonsaponifiable mat,erial was accomplished as follows. The petroleum ether extract of nonsaponifiable material (15 ml) was concentrated to dryness (water bath, 50") under h'z. The residue was dissolved in 0.5 ml of petroleum ether and gas chromatographed on lOyo silicone gum rubber (SE-30) on Chromosorb W (800-100 mesh), 4 i t X 0.26 in. glass column with the column temperature 240", detect,or temperature 285', injection port temperature 355', inlet pressure of 2.8 kgjcm2, and carrier gas (He) flow rate 50 ml/min; t,his gave a retention time for squalene of 13.0 min and for cholesterol, 24.4 min. Separation of the nonsaponifiable extract into squalene and cholesterol was accomplished by the met,hod of Langdon and Bloch.ll The petroleum ether extract (15 ml) was concentrated to dryness in a water bath (50') under Nz. The residue was chromatographed on acid-washed aluminal2 using petroleum ether (60-70"). The squalene fraction was checked for purity by glpc. Sterols were then eluted with MezCO-EtzO (1:l v/v). The cholesterol fraction was similarly checked for purity using glpc. Squalene- and cholesterol-containing eluates were concentrated to dryness in a water bath (50") under NZin standard counting vials. PPO solution (10 ml) was added and the radioactivity was determined with a Packard Tri-Carb liquid scintillat.ion counter.
played by the free constituent amino acids.2 In terpretatioii of the results of such studies is complicated by the hydrolysis of the peptides to form free amilio acids as often occurs in biological systems. In an attempt to produce a peptide analog whose study wocld not be complicated by hydrolysis, 2-benzyl-5-aminolevulinic acid was synthesized. 2-Benzyl-5-aminolevulinic acid (I) can be viewed as a n analog of glycylphenylalanine wherein the labile CONH group of the peptide has been replaced by the nonlabile COCHz group. All compounds used in biological testing were of the racemic form. 2-Benzyl-5-aminolevulinic acid was tested for its effect upon the growth of Escherichia coli 9723 and Leuconostoc mesenteroides P-60. Growth of E . coli was not inhibited by I when tested in the basal salts-glucose medium. However, as shomi in Table I,
Acknowledgment.-We are grateful to the ,Rational Institutes of Health for support of this work through Grant H E 12740-01.
I may either stimulate or inhibit the growth of E. coli
(10) T h e F a n d M Model 402 biomedical gas chromatograph equipped with flame ionization detector was employed in these studies. (11) R. G. Langdon and K. Bloch, J . B i d . Chem., 200, 129 (1953). (12) Alupharm Chemicals, Kew Orleans, La.
TABLE I EFFECTUPON GROWTHOF E . coli 9723a BY (BAL.4) IS MEDIA COKTAISIXG 8-2-T€[IESrL-DL-.1L.~SINE (TA4), GLYCYL-~-2-T€~IENYL-DL-.ILdSISE (GTA), GLYCYLGYLCYL-~-2-T€[IEKYL-DL-.kL.iNIiYE (G?T.I), A N D GYLCYtDGP€lESYLhLANINE(GPA)
2 - B E S Z Y L j - . I M I N O L E ~ U L I ~ I CA C I D
of dry wt of cells/ml of cultureSupplements
m -g BALB, mpmolesiml
0 20 60 200 600
TAb
GTAC
0
0
0 0
0.08
c. SL'VIITH'~ARID
0.20
TA, GTA, GPA'
0.13 0.12 0.10 0.06 0.01
0 0
a Incubated 15 hr a t 37". Medium contained 2 mpmoles of Medium TA/ml. Medium contained 6 mpmoles of GTA/ml. contained 6 mpmoles of GzTA/ml. e Jledium contained 200 mpmoles of TA, 200 mpmoles of GTA, and 20 mpmoles of GPA/ml.
depending upon the way the medium is supplemented with peptides of P-2-thienylalanine or phenylalanine. CO Et
+
C1- HJNCH-COCH2CHCOOH
I
8€lERI
